Gene therapy is a promising approach for treating hemophilia. Currently, their therapeutic potential is limited by the lack of safe and efficient delivery vectors. Although viruses remain the vectors of choice to achieve high efficiency of DNA transfer in vivo, reservation on their long-term safety lingers. Issues of immunogenicity and toxicity remain a challenge. Limitations of cell mitosis for retrovirus, contamination of adenovirus, and packaging constraints of adeno-associated virus also lessen their appeal. Consequently, non-viral vectors receive increasing attention on basis of ease of synthesis, low immunogenicity, and unrestricted plasmid size. Non-viral vectors also face less of a challenge in addressing pharmaceutical issues such as scale-up, storage stability, and quality control. However, the gene expression level achievable by non-viral gene delivery is low and transient. As a result, frequent administration is necessary to reach therapeutic level for gene medicine applications. To date virtually all non- viral transgene delivery has been either intravenous or intramuscular injection, which being invasive is unattractive and impractical for widespread use. In hemophilia gene therapy such an approach is less attractive and not much better than the current protein replacement therapy. Oral delivery may render gene therapy practical. The objective of this proposal is to test the hypothesis that the FVIII gene can be effectively delivered through the oral route using chitosan and poly(phosphoester)s as the non-viral gene carriers. We have demonstrated that oral administration of DNA-nanoparticles synthesized by complexing plasmid DNA with chitosan, a natural biocompatible polysaccharide, resulted in transduced gene expression in the intestinal epithelium. However, the transgene expression has been low in these studies. The objective of this proposal is to improve the nonviral oral gene delivery technology through optimization of carrier design and macroformulation for oral delivery. To define the potential of this oral gene medicine concept, we will pursue the following specific aims: 1) Design and synthesize gene carriers with characteristics favorable for oral delivery;2) Identify the important characteristics of DNA nanoparticles for optimal uptake;and 3) Evaluate the oral gene delivery systems in a FVIII-knock-out murine model. By proposing a focused approach to develop an effective oral gene medicine technology, we aspire to help realize the potential of gene therapy, that of applying gene as a drug. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to test the hypothesis that nonviral gene vectors can be effectively delivered through the oral route using chitosan and poly(phosphoester)s as the gene carriers. If successful, the oral nonviral gene delivery technology may have the potential to transform the healthcare for protein replacement therapies such as chronic anemia, growth hormone deficiency and diabetes mellitus. To define the potential of this oral gene medicine concept, we will pursue the following specific aims: 1) Design and synthesize gene carriers with characteristics favorable for oral delivery;2) Identify the important characteristics of DNA nanoparticles for optimal uptake;and 3) Evaluate the oral gene delivery systems in a FVIII- knock-out murine model.